Cytotoxicity of polymers intended for the extrusion-based additive manufacturing of surgical guides

Extrusion-based printing enables simplified and economic manufacturing of surgical guides for oral implant placement. Therefore, the cytotoxicity of a biocopolyester (BE) and a polypropylene (PP), intended for the fused filament fabrication of surgical guides was evaluated. For comparison, a medically certified resin based on methacrylic esters (ME) was printed by stereolithography (n = 18 each group). Human gingival keratinocytes (HGK) were exposed to eluates of the tested materials and an impedance measurement and a tetrazolium assay (MTT) were performed. Modulations in gene expression were analyzed by quantitative PCR. One-way ANOVA with post-hoc Tukey tests were applied. None of the materials exceeded the threshold for cytotoxicity (< 70% viability in MTT) according to ISO 10993-5:2009. The impedance-based cell indices for PP and BE, reflecting cell proliferation, showed little deviations from the control, while ME caused a reduction of up to 45% after 72 h. PCR analysis after 72 h revealed only marginal modulations caused by BE while PP induced a down-regulation of genes encoding for inflammation and apoptosis (p < 0.05). In contrast, the 72 h ME eluate caused an up-regulation of these genes (p < 0.01). All evaluated materials can be considered biocompatible in vitro for short-term application. However, long-term contact to ME might induce (pro-)apoptotic/(pro-)inflammatory responses in HGK.

are commonly used for material extrusion, while polyolefins are rarely applied for extrusion-based printing, although they are widely used for plastic production also for medical applications 13 . These high molar mass hydrocarbons are chemically resistant and mechanically durable due to their semi-crystalline properties and are able to undergo steam sterilization as a result of their high Vicat softening temperature [16][17][18] . Therefore, polyolefins such as polypropylene (PP) may provide a further cost-effective alternative for the production of accurate sterilizable surgical guides with well-balanced property profiles. When applied in the form of 3D printed surgical guides, polymers come into direct contact with intraoral hard and soft tissue cells. Monomer release was described from resins intended for the vat photopolymerization of surgical guides 19 and occlusal splints 20 while several studies mention possible adverse effects of resin-based materials in the oral environment [21][22][23] . Investigations regarding the potential cytotoxicity of substances eluted from extruded materials on human oral cells are sparse. Therefore, a biological risk assessment of BE and PP prior to their intraoral application is mandatory to evaluate potential cytotoxic effects on human tissue-specific cells.
The present study aimed to evaluate the in vitro biocompatibility of the two processed filaments, BE and PP, by using human gingival keratinocytes (HGK) according to the ISO guidelines 10993-5:2009 24 and 10993-12:2021 25 . As a comparison, a commercially available photopolymerizable resin based on methacrylic-esters (ME) and approved for vat photopolymerization of medical devices (class 1) was used. In order to investigate potential cytotoxic effects, interleukin 1β (IL1B), interleukin 6 (IL6) and tumor necrosis factor (TNF) encoding for inflammation in HGK were investigated. Genes under study included annexin A5 (ANAX5), Caspase 8 (CASP8), and Caspase 9 (CASP9) as representatives of (pro-)inflammation in HGK. The null hypothesis assumed no differences in terms of biocompatibility between BE, PP, and ME.

Material and methods
Investigational materials. An experimental commercially available biocopolyester filament with a diameter of 1.75 mm (BE; GreenTec Pro, Extrudr, Lauterbach, Austria) based on a polylactic acid-based blend with improved temperature resistance and compostability according to EN 13432:2002 26 was investigated. In addition, a semi-crystalline polypropylene (PP) filament was extruded from a medical-grade (United States Pharmacopeia (USP) Class VI) injection molding granulate (Healthcare PP, Purell type, LyondellBasell Industries B.V., Rotterdam, The Netherlands) 27 and examined. The PP filament was fabricated using a twin-screw extruder (Teach-Line™, ZK 25 T, Collin, Ebersberg, Germany) at 180 °C and 45 rpm, with a 3.3 mm die diameter, a watercooling system, and a winding unit (take-off speed 90 mm s −1 ). The resulting filament cross-section exhibited dimensions of 2.8 × 2.6 (± 0.05) mm. The dimensions were determined with a micrometer screw at 50 randomly selected points along the filament. For comparison, a commercially available medical Class I photopolymerizable resin (ISO 10993-1:2009 and USP Class VI) based on methacrylic esters (ME; Dental SG Resin, Formlabs, Boston, MA, USA) and intended for the manufacture of surgical guides by SLA was used. Detailed information regarding the composition of the materials are summarized in Table 1.

Preparation of samples and eluates.
A standardized test specimen (15 × 15 × 3 mm) was designed using a Computer-Aided Design (CAD) software (Meshmixer 3.5, Autodesk, Inc., San Rafael, CA, USA) and imported as a standard tessellation language (STL) file into the corresponding nesting software. Thereafter, 18 samples were printed for each of the three groups (PP, BE, and ME) according to ISO 10993-12:2021 25 which specifies the requirements for sample preparation. Extrusion of both experimental filaments (PP, BE) was performed at a printing speed of 50 mm s −1 , layer height of 0.2 mm with 100% infill. AM of PP was conducted at a nozzle temperature of 210 °C and a build plate temperature of 60 °C (Ultimaker S5, Ultimaker B.V., The Netherlands). Specimens of group BE were printed at a nozzle temperature of 220 °C and a build plate temperature of 60 °C (Prusa i3, MK3, Prague, Czech Republic). Two different extrusion-based printers were used for the manufacturing of the samples since they were produced at two different locations. Samples of group ME were produced with a layer thickness of 0.05 mm using an SLA printer (Form 2, Formlabs, Boston, MA, USA) and subsequently post-processed with rinsing in 99% isopropanol for 5 min, air drying and light-curing for 30 min at 60 °C (λ = 405 nm). Finally, support structures were removed. Samples were cleaned in 75% ethanol for 5 min and rinsed with water before being processed in a washer-disinfector (PG 8536, Miele, Gütersloh, Germany) containing a liquid detergent (neodisher MediClean forte, Dr. Weigert AG, Zug, Switzerland). All specimens were steam sterilized in an autoclave (Webeco, Series EC, Selmsdorf, Germany) at 134 °C for 5 min. For the preparation of the eluates, each sample was immersed in 5 mL keratinocyte growth medium (KG-M1, PromoCell, Heidelberg, Germany) in six well plates (Greiner Bio-One, Frickenhausen, Germany). One-half of the samples was stored at 37 °C, 97% humidity, and 5% CO 2 for 24 h and the other half for 72 h. All eluates were kept at 4 °C and absence of light before application to cell cultures. Impedance measurement. According to the manufacturer´s instructions, the impedance was measured with a real-time cell analyzing system (RTCA) (iCelligence, ACEA Biosciences, Inc., San Diego, CA, USA). Before adding the cells, 100 µl of cell medium containing the eluates (and without for the control) were applied to each of the eight wells of the E-plate E8 of the RTCA system, and a background impedance measurement without HGK was taken. Subsequently, 400 µl medium were applied to each of the wells containing HGK to achieve a density of 1.1 × 10 4 cells/cm 2 . Plates were incubated with standard cell culture conditions (37 °C; 97% humidity; 5% CO 2 ) for 5 d. RTCA iCelligence software (ACEA Biosciences) was used for data acquisition and analysis. Tests were conducted with three biological and two technical replicates for each of the evaluated polymers. The cell index (CI) describes the result of the impedance induced by adherent cells to the electron flow and was normalized at the time point of substance addition.

Colorimetric tetrazolium assay (MTT). A quantitative colorimetric assay with tetrazolium salt
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid; MTT) (Abcam, Cambridge, UK) was performed in accordance to the ISO guideline 10993-5:2009 24 to detect the viability of HGK after incubation with the eluates. This assay is based on the reduction of yellow water-soluble MTT in living cells to blue-violet insoluble formazan. To conduct the MTT assay, 200 µl medium containing HGK were applied to each well of a 96 well plate (Greiner Bio-One, Frickenhausen, Germany) to obtain a density of 1.4 × 10 4 cells/cm 2 . Subsequently, the plates were incubated at 37 °C for 24 h to allow cell adherence. Cell culture medium was then replaced with the one containing the eluates and cells were re-incubated at previously described conditions. After removing the eluate medium, 100 µl MTT solution was added to the cell cultures and incubated for 3 h. Finally, to measure the formazan concentration, a scanning spectrophotometer (Infinite 200 PRO, Tecan, Männedorf, Switzerland) with a wavelength of 590 nm and the compatible data acquisition software were used.

Results
Impedance measurement. A real-time impedance measurement was performed to monitor live cell proliferation, adhesion, and viability of HGK after exposure to the eluates as determined by the CI (Fig. 1). After an incubation period of 24 h, a reduced CI was observed for the 72 h eluates of ME (12% reduction) compared to the untreated control (p < 0.05) (Fig. 2a). None of the evaluated 24 h eluates showed a significant regulation of the CI compared to the control after an incubation period of 24 h (p > 0.05). After an incubation period of 72 h, both ME eluates (24 h and 72 h eluate) showed a significant inferior CI compared to the control (32% reduction    (Fig. 3): The cell number was distinctly reduced and the cell morphology was partially apoptotic after incubation with ME (both eluates) for 72 h. In contrast, HGK revealed a confluent cell lawn after incubation with PP and BE for 72 h which had a similar appearance as after incubation with the non-treated control (Fig. 3a,b,d). These observations were consistent with the results of the impedance measurement for PP, L, and the control.
MTT assay. The MTT assay showed a viability of 78% after incubation of HGK with the 24 h PP eluate (Fig. 4). The viability was reduced to 84% after incubation with the 72 h BE eluate. Both ME eluates as well as the 72 h PP and the 24 h BE eluate showed no reduced viability in the MTT assay. In accordance with ISO guideline 24 , a test specimen exhibits a cytotoxic potential when the viability is reduced to < 70%, which occurred for none of the evaluated polymers or time points.

Gene expression analysis.
A qRT-PCR analysis was performed to investigate possible modulations of relevant genes related to apoptosis and inflammation. Electropherograms and virtual gels (Fig. 5) obtained from the total RNA extraction of HGK, both the unexposed control and after incubation with eluates, showed two distinct peaks for the control 18S and 28S rRNAs. The integrity and amount of total RNA was measured in all groups and considered as appropriate for the qRT-PCR analysis. Figure 6 shows the results of the qRT-PCR  The biomarkers expression of (pro-)apoptosis ANXA5, CASP8, and CASP9 resulted in a significant upregulation after incubation with the ME 72 h eluate compared to the control (p < 0.05). In contrast, no significant regulation was observed after incubation with the 24 h eluate (p > 0.05). The 24 h eluates of the extrusion-based printed specimens PP and BE showed no up-regulation of the respective biomarkers (p > 0.05). After incubation with the 72 h PP eluate, a down-modulation of the biomarkers ANXA5 and CASP8 were observed (p < 0.05). Comparing the eluate time points for each group, significant differences were found for PP and ME for the biomarkers ANXA5, CASP8, and CASP9 (p < 0.05). No significant modulations were observed for group BE between the two evaluated eluate time points for all investigated biomarkers (p > 0.05).
HGK showed an increase of the inflammatory-related biomarker IL1B after incubation with the 72 h ME eluate (p = 0.01) compared to the non-treated control. In contrast, the 24 h PP eluate caused a significant downregulation for IL1B (p < 0.05). The other tested eluates exhibited a tendency toward down-regulation regarding this inflammation marker. After incubation with the 24 h ME eluate, HGK showed an increased expression of the biomarker interleukin 6 (IL6) (p < 0.01) in comparison to the untreated control, whereas the 72 h eluate of this polymer caused a tendency toward down-regulation. No significant modulations (p > 0.05) compared to the control were observed for IL6 after incubation with the remaining groups. In addition, the biomarker TNF Figure 6. Normalized up-/down-modulation of selected genes of apoptosis (a) and inflammation (b) in HGK exposed to 24 h and 72 h eluates from PP, BE and ME for 72 h referring to the untreated control. Data are normalized to the untreated control cells and the unregulated housekeeping genes GAPDH, RPL13A and UBC. The normalized expression was calculated using the 2 −ΔΔCT method 29 . Asterisks (*) indicate significance (p < 0.05) compared to the untreated cells. www.nature.com/scientificreports/ showed no significantly modulated gene expression compared to the control group for the evaluated materials and eluate time points (p > 0.05). When comparing 24 h with 72 h eluate time points of group PP, significant differences were observed for the genes IL6 and TNF (p < 0.05). Group ME showed significant differences between the two eluate time points for IL1B and IL6 (p < 0.05), whereas BE revealed no significant modulations in gene expression of the inflammatory biomarkers between the two eluate times (p > 0.05).

Discussion
Numerous materials for resin-based AM of surgical guides are available on the market but the demand for inexpensive and sustainable materials as well as for simple fabrication workflows is increasing 30 . The objective of the present study was to evaluate the in vitro biocompatibility of two experimental polymers intended for the AM of surgical guides by material extrusion in comparison to a certified light-curable resin used for vat photopolymerization of medical class I surgical guides (ISO 10993-5:2009 and USP Class VI). A commonly used biopolymer for material extrusion in the non-medical field is based on PLA 31 . However, pure PLA is prone to deformation when steam sterilized at temperatures above 121 °C. Therefore, an experimental biocopolyester blend based on PLA, other biopolymers, and calcium carbonate fillers was utilized for optimized temperature resistance. In addition, a semi-crystalline PP was included in the present biocompatibility evaluation. PP belongs to the group of polyolefins which are high-molecular hydrocarbon materials derived from fossil or renewable energy sources 32 . Due to its semi-crystalline nature, PP appears chemically resistant and withstands steam sterilization. A PP injection molding granulate with certified biocompatibility according to USP class VI was used for the extrusion of the PP filament 27 . However, material properties might suffer from alteration during the extrusion or printing process leading to potential cytotoxic effects. The null hypothesis of this study assumed no differences in terms of biocompatibility on HGK between the evaluated materials. HGK were selected based on their anatomical location on the surface of the oral mucosa. Due to the in-vitro short lifetime of primary HGK with resulting limited reproducibility of the experiments, immortalized HGK were chosen. Nevertheless, the differentiation pattern of this cell line is well-preserved, making HGK comparable to the original tissue and particularly suitable for experiments on modulations of gene expression 28 . The eluates were prepared according to ISO guideline 10993-12:2021, which describes sample preparation and reference materials. Furthermore, the incubation time of 24 h for the MTT assay is defined in ISO standard 10993-5:2009 while the incubation period of 72 h for the qRT-PCR was based on the results of the impedance measurement. The extended incubation period was also used to evaluate materials for long-term intraoral application, such as splints or aligners 33 .
RTCA iCelligence™ and MTT assay were used to measure the proliferation and viability of HGK after incubation with the eluates. None of the eluates showed a reduced viability (threshold set at < 70%) in the MTT assay according to ISO 10993-5:2009, which was in accordance with the RTCA measurement and cells' morphology after incubation with eluates of both filaments, indicating no cytotoxic effects for PP and L. Solely the 72 h BE eluate showed a slightly reduced CI after several days of incubation. A significantly reduced CI was observed for both ME eluates (24 and 72 h), with HGK also showing a significantly reduced cell density and increased apoptotic appearance under the light microscope. These results did not occur in the MTT assay which might suggest a higher specificity and sensitivity of the RTCA system 34 . A further advantage of the RTCA system is the comprehensive documentation of cellular proliferation, growth, and morphological chances (not only in terms of viability) over the entire period of the investigation, rather than being limited to individual endpoints. In addition, no formazan products are generated as in the MTT assay (which might interact with the compounds of the eluates and affect the final results) 35,36 .
To investigate the influence of the eluates on gene expression level of HGK, a qRT-PCR analysis was performed. Potential cytotoxic effects, which might have led to a reduced CI and morphological changes of HGK after incubation with ME for an extended period, were investigated based on relevant biomarkers for (pro-) apoptosis and (pro-)inflammation after 72 h of incubation. The evaluated caspases CASP8 and CASP9 are initiator caspases which can induce apoptosis 37,38 . ANXA5 binds efficiently to phosphatidylserine and is transported to the outer surface of the plasma membrane in the early apoptosis stage 39 . Furthermore, IL1B is secreted from keratinocytes and fibroblasts and represents a marker for the early inflammatory response 40,41 . The presence of IL6 in the inflamed tissue also plays a decisive role in the onset and maintenance of periodontal inflammatory conditions such as periodontitis and gingivitis 42 . The additionally investigated marker TNF is a pro-inflammatory cytokine associated to tissue degeneration and able to stimulate osteoclastic activity 43 . In the present study, an up-regulation of the early apoptosis biomarkers ANXA5, CASP8 and CASP9 was observed after incubation with the 72 h ME eluate. Since these modulations did not occur for the 24 h eluate, it can be assumed that the release of cytotoxic compounds occurs after a longer period. Polydorou et al. 44 showed that light-cured composite materials release monomers over a time period of up to 1 year, which might have adverse effects on oral tissues. Released monomers, such as bisphenol A-glycidyl methacrylate (BisGMA) of a methacrylate-based nanohybrid composite resin contained in a related form in the investigated SLA-resin, induced severe apoptosis in HGK after 4 d 45 . Moreover, in human gingival fibroblasts (HGF) apoptosis induced by oxidative stresses was observed after the release of monomers 22,46 . The tendency toward cytotoxic effects on gene expression level after incubation with the 72 h ME eluates was corroborated with an up-regulation of IL1B and IL6, with the latter biomarker being regulated by the 24 h eluate. Therefore, the present results suggest that monomers can be released from ME test specimens after an extended period despite elaborated washing and light-curing steps and may induce (pro-)apoptotic/(pro-)inflammatory modulations in HGK. The null hypothesis assuming no differences in cytotoxicity between the evaluated materials was therefore rejected. Resins for vat photopolymerization still contain numerous compounds and photoinitiators in the processed parts that are cytotoxic 47 and may induce (pro-)apoptotic/(pro-)inflammatory modulations in HGK. This outcome seems in agreement with a similar www.nature.com/scientificreports/ study, which assessed low cytotoxic effects of the evaluated SLA-resin in mesenchymal stem cells and showed significantly decreased viability in the MTT assay after an extended incubation period, although the printed resin is classified as biocompatible 21 . Zhu et al. questioned the safety of several resins for vat photopolymerization in terms of release of toxic substances in water-based cell culture media 48 . As in our study, cytotoxic effects in form of mortality of aquatic bioindicators were not observed after 24 h but increased dramatically over a prolonged period of up to 72 h 48 . The inconsistencies between official certifications and the observed results might derive from the different incubation periods. PLA and ABS for extrusion-based AM seem not to induce behavioral abnormalities in zebrafish compared to the evaluated photopolymerizable resins 48 , which is in agreement with our results on gene expression level. None of the extrusion-based materials (PP and BE) showed an up-regulation of the evaluated (pro-)apoptotic/ (pro-)inflammatory biomarkers. The significant down-regulation of ANXA5 and CASP8 caused by the PP 72 h eluate might even indicate an anti-apoptotic side effect on HGK. These results were enhanced by the activity of the investigated inflammatory markers IL1B, IL6, and TNF which tended toward down-regulation after incubation with PP. These properties are known for polyolefins, which are rarely processed by material extrusion, but are widely used for various medical applications like e.g., tubes, syringes and piezoelectric materials for their high purity and low allergy potential 32,49 . The fact that BE did not show limitations on gene expression level with regard to its biocompatibility is consistent with the literature 50,51 . No adverse side effects in terms of biocompatibility for HGK were observed after extrusion-based AM and steam sterilization. This can be attributed to a different chemical structure and the absence of photoinitiators or monomers in filaments intended for material extrusion, differently than for photo-curable 3D printing materials 19 . Nevertheless, further studies evaluating other cell cultures as e.g. osteoblasts, are needed. An assessment of cells in the tissue compound, such as by means of interactive cell systems of HGK and HGF, would be necessary to mimic similar in-vivo conditions 28 .

Conclusions
According to the results of this study, all evaluated materials can be considered as biocompatible in vitro for short-term intraoral usage as for guided implant positioning. Furthermore, both experimental filaments showed reduced (pro-)apoptotic/(pro-)inflammatory modulations compared to the investigated SLA-resin. Material extrusion of surgical guides made of PP and BE thus might represent a cost-effective alternative to currently used materials and manufacturing processes.

Data availability
All data generated or analysed during this study are included in this published article.